Acute myeloid leukemia (AML) is the most common type of leukemia in adults and has poor overall outcome because of therapy resistance and subsequent relapse. Leukemia stem cells (LSCs) that can initiate and maintain AML are considered potential sources of relapse because they are not effectively eliminated by standard chemotherapy. Our long-term goals are to define the molecular mechanism(s) regulating LSC generation and maintenance, and to identify novel mechanism-based LSC targeted therapies. One of the most common chromosome aberrations found in AML patients is inversion of chromosome 16, which creates the fusion gene CBFb-MYH11 that encodes the CBF-SMMHC (CM) fusion protein. The objectives of this application are to define how histone deacetylase 8 (HDAC8) contributes to CM-induced AML pathogenesis, define the molecular mechanism(s) underlying HDAC8 activity and evaluate the efficacy of isoform-selective inhibitors (HDAC8i) in targeting human AML LSCs. Our central hypothesis is that HDAC8 activity is deregulated through both inv(16)-dependent and -independent mechanisms, and that aberrant HDAC8 activity contributes to AML LSC transformation and maintenance by disrupting p53 acetylation. Therefore, inhibiting HDAC8 by using HDAC8i may reactivate p53 and enhance targeting of AML LSCs. The specific aims are: 1) to define how HDAC8 contributes to inv(16)-induced AML pathogenesis and LSC maintenance, and 2) to determine the mechanism(s) underlying HDAC8 activity and the efficacy of HDAC8 isoform-selective pharmacological inhibitors in targeting non-inv(16) AML LSCs. In Specific Aim 1, we will define how CM alters HDAC8 and p53 activities; determine the role of HDAC8 in CM-induced LSC transformation and maintenance by using our conditional CM knock-in mice that also carry a new conditional Hdac8 deletion allele; and validate the findings in primary AML LSCs from inv(16)+ patients. In Specific Aim 2, we will determine the correlation between HDAC8 expression and levels of SOX4 and HOXA9 or p53 status in non-inv(16) AML LSCs; determine the functional consequences of HDAC8 overexpression and whether the HOXA9/SOX4 axis contributes to HDAC8 deregulation; and evaluate the effects of HDAC8i on p53 activity, LSC proliferation and resistance to therapy. To evaluate the function of HDAC8, we have generated mice carrying a new conditional Hdac8 deletion allele. In addition, we will use a combination of shRNA-mediated knockdown and novel pharmacological HDAC8i. The proposed studies are expected to establish molecular mechanism(s) underlying HDAC8 deregulation, as well as a novel HDAC8-mediated post-translational p53 inactivating mechanism in AML pathogenesis. In addition, these studies will reveal mechanisms of action and whether HDAC8i can reactivate p53 and enhance elimination of AML LSCs. The results will offer novel rationales and novel HDAC8 isoform-selective pharmacological inhibitors for targeted therapy, and will have translational implications for AML, as well as other cancer types in which p53 is not mutated.